Experimentelle Untersuchungen an einem Massenauftreten von neotenen Triton taeniatus

Ohne Zusammenfassung     

Reinigung und serologischer Nachweis des Erbsenenationenmosaik-Virus (pea enation mosaic virus)

Trotzdem von einer zunehmenden Bedeutung der Schneeschimmelkrankheit, hervorgerufen durch den Pilz Microdochium nivale, berichtet wird, liegen nur wenige Information über die Physiologie der Schadwirkung dieses Pilzes, insbesondere bei Blattbefall vor. Daher werden in dieser Arbeit die Einflüsse einer Inokulation mit M. nivale auf die Stickstoffaufnahme, ‐Verteilung sowie die source‐sink‐Beziehungen der Wirtspflanze untersucht. Die Weizenpflanzen wuchsen in Glaskabinen oder im Rhizotron unter Semifreilandbedingungen bzw. in Klimakammern. Durch Gabe von ¹⁵NH₄ ¹⁵NO₃ (2, 5 at‐% ¹⁵N exc.) zur Inokulation des Saatgutes, der Ähren oder verschieden inserierten Blättern wurden die Wirkungen der Krankheit auf den Stickstoffhaushalt des Weizens untersucht. Wenn keine zusätzliche Beeinflussung durch andere, biotische oder abiotische Streßfaktoren erfolgte, waren weder die Stickstoffaufnahme durch die Wurzeln, der Transport in die Körner, der Einbau in diese noch die Remobilisierung und Retranslokation zuvor gespeicherter N‐Verbindungen durch die Inokulation gehemmt. In einigen Versuchen war der Stickstoffhaushalt sogar stimuliert; die Stickstoffaufnahme, der Transport und Einbau in die Körner waren in den befallenen Pflanzen bis zur Reife höher als in den gesunden Pflanzen. Die Folgen dieser Wirkungen für den Kohlen‐hydrathaushalt und die Beteiligung von Phytohormonen an diesen Veränderungen werden diskutiert.     

Ultramicroscopy Reveals Axonal Transport Impairments in Cortical Motor Neurons at Prion Disease

The functional imaging of neuronal circuits of the central nervous system is crucial for phenotype screenings or investigations of defects in neurodegenerative disorders. Current techniques yield either low penetration depth, yield poor resolution, or are restricted by the age of the animals. Here, we present a novel ultramicroscopy protocol for fluorescence imaging and three-dimensional reconstruction in the central nervous system of adult mice. In combination with tracing as a functional assay for axonal transport, retrogradely labeled descending motor neurons were visualized with >4 mm penetration depth. The analysis of the motor cortex shortly before the onset of clinical prion disease revealed that >80% neurons have functional impairments in axonal transport. Our study provides evidence that prion disease is associated with severe axonal transport defects in the cortical motor neurons and suggests a novel mechanism for prion-mediated neurodegeneration.     

Isochorismate hydroxymutase from Rubiaceae cell suspension cultures

The enzyme catalysing the isomerisation of chorismic to isochorismic acid (isochorismate hydroxymutase E.C. 5.4.99.6) has been detected in protein preparations of various cell suspension cultures derived from plants of Rubiaceae species.Abbreviations OSB o-Succinylbenzoic acid - Tris Tris(hydroxymethyl)aminomethane - DEAE Diethylaminoethyl cellulose     

Epicocconone staining: A powerful loading control for Western blots

Western blot analysis is routinely employed for quantifying differences in protein levels between samples. To control equal loading and to arithmetically compensate loading differences, immunodetection of housekeeping proteins is commonly used. Due to potential biases, this approach has been criticized. Here, we evaluate epicocconone‐based total protein staining (E‐ToPS) as an alternative. We compared it with two other total protein stainings (Coomassie and Sypro Ruby) and with immunodetection of housekeeping proteins (β‐tubulin and glyceraldehyde 3‐phosphate dehydrogenase). Evaluation comprised both the natural and the synthetic epicocconone compound. Both compounds produced highly congruent results and showed more sensitive (≤ 1 μg) and less variable staining properties than the other variants. The high sensitivity of E‐ToPS, covering minute protein amounts, makes it a powerful loading control, especially for precious samples. Regarding biological and technical variances, E‐ToPS outperformed immunostaining against β‐tubulin and glyceraldehyde 3‐phosphate dehydrogenase. Furthermore, E‐ToPS had no impact on subsequent immunodetection, allowing for an early control of proper loading prior to immunodetection. In contrast to earlier studies, we found logarithmic staining properties for E‐ToPS, which should be considered when using it for arithmetic normalization. In conclusion, we demonstrate the superior power of E‐ToPS as a loading control for Western blots.     

Direct introduction of gene constructs into the pronucleus-like structure of cloned embryos: a new strategy for the generation of genetically modified pigs

Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed “nuclear injection”. To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.